GnRH antagonists V

ABSTRACT

Peptides which inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads, which can be used for therapeutic treatment. The peptides have the structure: 
     X-R 1  -R 2  -R 3  -Ser-Arg-R 6  -R 7  -Arg-Pro-R 10  wherein X is hydrogen, Acr or Ac; R 1  is β-D-2NAL, dehydroPro or 4Cl-D-Phe; R 2  is Cl-D-Phe, F-D-Phe, NO 2  -D-Phe, Br-D-Phe or C a  Me/Cl-D-Phe; R 3  is D-Trp or β-D-2NAL; R 6  is D-Tyr, β-D-2NAL or D-Arg; R 7  is Leu or NML; and R 10  is Gly-NH 2 , NHCH 2  CH 3  or D-Ala-NH 2  ; provided however that R 1  or R 3  or both are β-D-2NAL.

This invention was made with Government support under Contract Nos. NO 1-HD-2-2808 and NO 1-HD-4-2833 and under Grant No. HD-13527, awarded by the National Institutes of Health. The Government has certain rights in this invention.

This application is a continuation-in-part of pending application Ser. No. 556,148, filed Nov. 29, 1983 now U.S. Pat. No. 4,547,370.

The present invention relates to peptides which inhibit the release of gonadotropins by the pituitary gland in mammalians, including humans, and to methods of preventing ovulation and/or inhibiting the release of steroids. More particularly, the present invention is directed to peptides which inhibit gonadal function and the release of the steroidal hormones, progesterone and testosterone.

BACKGROUND OF THE INVENTION

One of the hypothalamic hormones acts as a factor that triggers the release of the gonadotropic hormones, particularly LH, and this hormone is referred to herein as GnRH although it has also been referred to as LH-RH and as LRF. GnRH has been isolated and characterized as a decapeptide having the following structure:

    p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2

Peptides are compounds which contain two or more amino acids in which the carboxyl group of one acid is linked to the amino group of the other acid. The formula for GnRH, as represented above, is in accordance with conventional representation of peptides where the amino group appears to the left and the carboxyl group to the right. The position of the amino acid residue is identified by numbering the amino acid residues from left to right. In the case of GnRH, the hydroxyl portion of the carboxyl group of glycine has been replaced with an amino group (NH₂). The abbreviations for the individual amino acid residues above are conventional and are based on the trivial name of the amino acid, e.g. p-Glu is pyroglutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, Leu is leucine, Arg is arginine, Pro is proline, Phe is phenylalanine and Ala is alanine. These amino acids together with valine, isoleucine, threonine, lysine, aspartic acid, asparagine, glutamine, cysteine and methionine are generally considered to be the common, naturally occurring or protein-derived amino acids. Except for glycine, amino acids of the peptides of the invention are of the L-configuration unless noted otherwise.

There are reasons for desiring to prevent ovulation in female mammalians, and the administration of GnRH analogs that are antagonistic to the normal function of GnRH have been used to prevent ovulation. For this reason, analogs of GnRH which are antagonistic to GnRH are being investigated for their potential use as a contraceptive or for regulating conception period. Such antagonists have also been found useful to regulate the secretion of gonadotropins in male mammals and can be empolyed as male contraceptives. They may also be used to treat conditions which are dependent upon the presence of an excess of gonodatropins, such as endometriosis and certain types of tumors, particularly prostate tumors, which have been found to be dependent upon endocrine secretions. It is desired to provide peptides which are strongly antagonistic to endogenous GnRH and which prevent secretion of LH and the release of steroids by the gonads of mammals.

SUMMARY OF THE INVENTION

The present invention provides peptides which inhibit the release of gonadotropins in mammalians, including humans, and also provides methods for inhibiting the release of steroids by the gonads of male and female mammalians. The improved GnRH analogs are antagonistic to GnRH and have an inhibitory effect on the reproduction processes of mammalians. These analogs may be used to inhibit the production of gonadotropins and sex hormones under various circumstances including precocious puberty, hormone dependent neoplasia, dysmenorrhea and endometriosis.

Generally, in accordance with the present invention, peptides have been synthesized which strongly inhibit the secretion of gonadotropins by the pituitary gland of mammalians, including humans, and/or inhibit the release of steroids by the gonads. These peptides are analogs of GnRH wherein there is a 1-position substitution such as β-(2-naphthyl) D-alanine (hereinafter β-D-2NAL) or dehydroPro or 4Cl-D-Phe, a 3-position substitution such as D-Trp or β-D-2NAL, a 5-position substitution of Arg for Tyr, a 6-position D-isomer substitution as generally known in the art and a specific substituent in the 2-position. The 1-position substituent may be modified so that its alpha-amino group contains acetyl(Ac) or acrylyl(Acr), or it may be unmodified. Modified D-Phe is present in the 2-position and provides increased antagonistic activity as a result of the specific modifications present in the benzene ring, particularly when the alpha carbon atom is also methylated. A single substitution for hydrogen is made in the para- or 4-position, with chloro and fluoro being preferred. A double substitution in the residue is preferred, i.e., one on the alpha carbon atom and one in the benzene ring, e.g., C^(a) Me/4Cl-D-Phe. D-Tyr or D-Arg is preferably present in the 6-position. The substitutions in the 7- and 10-positions are optional.

Because these peptides are highly potent to inhibit release of LH, they are often referred to as GnRH antagonists. The peptides inhibit ovulation of female mammals when administered at very low levels at proestrous and are also effective to cause resorption of fertilized eggs if administered shortly after conception. These peptides are also effective for the contraceptive treatment of male mammals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

More specifically, the peptides of the present invention are represented by the following formula:

X-R₁ -R₂ -R₃ -Ser-Arg-R₆ -R₇ -Arg-Pro-R₁₀ wherein X is hydrogen, Acr or Ac; R₁ is β-D-2NAL, dehydroPro or 4Cl-D-Phe; R₂ is Cl-D-Phe, F-D-Phe, NO₂ -D-Phe, Br-D-Phe or C^(a) Me/Cl-D-Phe; R₃ is D-Trp or β-D-2NAL; R₆ is D-Tyr, β-D-2NAL or D-Arg; R₇ is Leu or NML; and R₁₀ is Gly-NH₂, NHCH₂ CH₃ or D-Ala-NH₂ ; provided however that either R₁ or R₃ or both are β-D-2NAL.

By β-D-NAL is meant the D-isomer of alanine which is substituted by naphthyl on the β-carbon atom, which may also be designated 3-D-NAL. Preferably β-D-2NAL is employed which means that the β-carbon atom is attached to naphthalene at the 2-position on the ring structure; however, β-D-1NAL is considered an equivalent. By dehydroPro is meant 3,4 dehydroproline. By NML is meant the methyl substitution on the alpha-amino group of Leu.

The peptides of the present invention can be synthesized by classical solution synthesis or by a solid phase technique using a chloromethylated resin, a methylbenzhydrylamine resin(MBHA) or a benzhydrylamine (BHA) resin. The solid phase synthesis is conducted in a manner to stepwise add the amino acids in the chain in the manner set forth in detail in the U.S. Pat. No. 4,211,693. Side-chain protecting groups, as are well known in the art, are preferably added to Ser, Tyr and Arg and may optionally be added to Trp, before these amino acids are coupled to the chain being built upon the resin. Such a method provides the fully protected intermediate peptidoresin.

The intermediates of the invention may be represented as:

X¹ -R₁ -R₂ -R₃ (X²)-Ser(X³)-Arg(X⁵)-R₆ (X⁴ or X⁵)-R₇ -Arg(X⁵)-Pro-X⁶ wherein: X¹ is an a-amino protecting group of the type known to be useful in the art in the stepwise synthesis of polypeptides and when X in the desired peptide composition is a particular acyl group, that group may be used as the protecting group. Among the classes of a-amino protecting groups covered by X¹ are (1) acyl-type protecting groups, such as formyl (For), trifluoroacetyl, phthalyl, p-toluenesulfonyl (Tos), benzoyl (Bz), benzensulfonyl, o-nitrophenylsulfenyl (Nps), tritylsulfenyl, o-nitrophenoxyacetyl, acrylyl (Acr), chloroacetyl, acetyl (Ac) and γ-chlorobutyryl; (2) aromatic urethan-type protecting groups, e.g., benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chloro-benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as tertbutyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6) alkyl-type protecting groups, such as allyl (Aly), triphenylmethyl(trityl) and benzyl (Bzl); (7) trialkylsilane groups, such as trimethylsilane. The preferred a-amino protecting group is Boc when X is hydrogen.

X² is hydrogen or a protecting group for the indole nitrogen, such as formyl or benzyl; however in many syntheses there is no need to protect Trp.

X³ is hydrogen or a protecting group for the alcoholic hydroxyl group of Ser and is selected from the group consisting of acetyl, benzoyl, tetrahydropyranyl, tert-butyl, trityl, benzyl and 2,6-dichlorobenzyl. Benzyl is preferred.

X⁴ is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, benzyl, benzyloxycarbonyl, 4-bromobenzyloxycarbonyl and 2,6-dichlorobenzyl. 2,6-dichlorobenzyl is preferred.

X⁵ is a protecting group for the nitrogen atoms of Arg and is selected from the group consisting of nitro, Tos, benzyloxycarbonyl, adamantyloxycarbonyl, and Boc; alternatively X⁵ may be hydrogen, which means there are no protecting groups on the side chain nitrogen atoms of arginine. Tos is preferred.

X⁶ is selected from the group consisting of Gly-O-CH₂ -[resin support]; O-CH₂ -[resin support]; Ala-O-CH₂ -[resin support]; Gly-NH-[resin support]; D-Ala-NH-[resin support]; and OH, ester, amide and hydrazide of either Gly or D-Ala or attached directly to Pro.

The criterion for selecting side chain protecting groups for X² -X⁵ is that the protecting group must be stable to the reagent under the reaction conditions selected for removing the a-amino protecting group at each step of the synthesis. The protecting group must not be split off under coupling conditions, and the protecting group must be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain.

When the X⁶ group is Gly-O-CH₂ -[resin support], D-Ala-O-CH₂ -[resin support] or O-CH₂ -[resin support], the ester moiety of one of the many functional groups of the polystyrene resin support is being represented. When the X⁶ group is Gly-NH-[resin support] or D-Ala-NH-[resin support], an amide bond connects Gly or D-Ala to BHA resin or to a MBHA resin.

When X is Ac or Acr in the final formula, it may be possible to employ it as the X¹ protecting group for the a-amino group of D-NAL by adding it before the coupling of this last amino acid to the peptide chain. However, a reaction is preferably carried out with the peptide on the resin (after deblocking the a-amino group while the side-chain groups remain protected), e.g. by reacting with acetic acid in the presence of dicyclohexyl carbodiimide (DCC) or preferably with acetic anhydride or by another suitable reaction as known in the art.

The fully protected peptide can be cleaved from a chloromethylated resin support by ammnolysis, as is well known in the art, to yield the fully protected amide interemediate. Deprotection of the peptide, as well as cleavage of the peptide from a benzhydrylamine resin, can take place at 0° C. with hydrofluoric acid (HF). Anisole is preferably added to the peptide prior to treatment with HF. After the removal of HF, under vacuum, the cleaved, deprotected peptide is conveniently treated with ether, decanted, taken-up in dilute acetic acid and lyophilized.

Purification of the peptide is effected by ion exchange chromatography on a CMC column, followed by partition chromatography using the elution system: n-butanol; 0.1N acetic acid (1:1 volume ratio) on a column packed with Sephadex G-25, or by using HPLC, as known in the art.

The invention thus also provides a method for making a peptide or a nontoxic salt thereof, which peptide has the formula: X-R₁ -R₂ -R₃ -Ser-Arg-R₆ -R₇ -Arg -Pro-R₁₀ wherein X is hydrogen, Acr or Ac; R₁ is β-D-2NAL, dehydroPro or 4Cl-D-Phe; R₂ is Cl-D-Phe, F-D-Phe, NO₂ -D-Phe, Br-D-Phe or C^(a) Me/Cl-D-Phe; R₃ is D-Trp or β-D-2NAL; R₆ is D-Tyr, β-D-2NAL or D-Arg; R₇ is Leu or NML; and R₁₀ is Gly-NH₂, NHCH₂ CH₃ or D-Ala-NH₂, which method comprises (a) forming an intermediate compound having the formula: X¹ -R₁ -R₂ -R₃ (X²)-Ser(X³)-Arg(X⁵)-R₆ (X⁴ or x⁵)-R₇ -Arg(X.sup. 5)-Pro-X⁶ wherein X¹ is hydrogen or an a-amino protecting group; X² is hydrogen or a protecting group for the indole nitrogen of Trp; X³ is hydrogen or a protecting group for the alcoholic hydroxyl group of Ser; X⁴ is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr; X⁵ is hydrogen or a protecting group for the nitrogen atoms of Arg; and X⁶ is selected from the group consisting of Gly-O-CH₂ -(resin support), O-CH₂ -(resin support), D-Ala-O-CH₂ -(resin support), Gly-NH-(resin support), D-Ala-NH-(resin support), Gly-NH₂, NHCH₂ CH₃ and esters, amides and hydrazides of Gly or D-Ala; (b) splitting off one or more of the groups X¹ to X⁵ and/or cleaving from any resin support included in X⁶ and, if desired, converting a resulting peptide into a nontoxic salt thereof.

The peptides of the invention are effective at levels of less than 100 micrograms per kilogram of body weight, when administered at about noon on the day of proestrous, to prevent ovulation in female rats. For prolonged suppression of ovulation, it may be necessary to use dosage levels in the range of from about 0.1 to about 2.5 milligrams per kilogram of body weight per day. These antagonists are also effective as contraceptives when administered to male mammals on a regular basis. Since these compounds will reduce testosterone levels (an undesired consequence in the normal, sexually active male), it may be reasonable to administer replacement dosages of testosterone along with the GnRH antagonist. These antagonists can also be used to regulate the production of gonadotropins and sex steroids for other purposes as indicated hereinbefore.

EXAMPLE

Peptides as indicated in TABLE I having the formula Ac-R₁ -R₂ -R₃ -Ser-Arg-R₆ -Leu-Arg-Pro-R₁₀ are prepared by the solid phase procedure referred to above.

                                      TABLE I                                      __________________________________________________________________________     R.sub.1  R.sub.2    R.sub.3                                                                              R.sub.6                                                                              R.sub.10                                       __________________________________________________________________________     1 4Cl--D-Phe                                                                            4Cl--D-Phe β-D-2NAL                                                                        D-Tyr D-Ala-NH.sub.2                                 2 "      4NO.sub.2 --D-Phe                                                                         "     D-Arg "                                              3 "      "          "     β-D-2NAL                                                                        "                                              4 dehydroPro                                                                            4Br--D-Phe "     D-Tyr NHCH.sub.2 CH.sub.3                            5 "      C.sup.a Me/4Cl--D-Phe                                                                     β-D-2NAL                                                                        β-D-2NAL                                                                        D-Ala-NH.sub.2                                 6 β-D-2NAL                                                                         "          D-Trp D-Tyr Gly-NH.sub.2                                   7 dehydroPro                                                                            4Cl--D-Ph  β-D-2NAL                                                                        "     NHCH.sub.2 CH.sub.3                            8 "      4F--D-Ph   "     "     D-Ala-NH.sub.2                                 9 "      4NO.sub.2 --D-Phe                                                                         "     "     Gly-NH.sub.2                                                                   (N MeLeu.sup.7)                                10                                                                               β-D-2NAL                                                                         4F--D-Phe  D-Trp "     NHCH.sub.2 CH.sub.3                            11                                                                               "      "          β-D-2NAL                                                                        β-D-2NAL                                                                        Gly-NH.sub.2                                   12                                                                               dehydroPro                                                                            "          "     "     "                                              13                                                                               β-D-2NAL                                                                         4Cl--D-Phe D-Trp "     D-Ala-NH.sub.2                                                                 (D-Arg.sup.5)                                  14                                                                               "      "          "     D-Arg D-Ala-NH.sub.2                                 15                                                                               4Cl--D-Phe                                                                            "          "     β-D-2NAL                                                                        "                                              __________________________________________________________________________

For purposes of an example, a representative solid phase synthesis of Peptide No. 1 above, which is referred to as [Ac-4Cl-D-Phe¹, 4Cl-D-Phe², β-D-2NAL³, Arg⁵, D-Tyr⁶, D-Ala¹⁰ ]-GnRH is set forth hereinafter. This peptide has the following formula: Ac-4ClD-Phe-4Cl-D-Phe-β-D-2NAL-Ser-Arg-D-Tyr-Leu-Arg-Pro-D-Ala-NH.sub.2

A BHA resin is used, and Boc-protected D-Ala is coupled to the resin over a 2-hour period in CH₂ Cl₂ using a 3-fold excess of Boc derivative and DCC as an activating reagent. The alanine residue attaches to the BHA residue by an amide bond.

Following the coupling of each amino acid residue, washing, deblocking and coupling of the next amino acid residue is carried out in accordance with the following schedule using an automated machine and beginning with about 5 grams of resin:

    ______________________________________                                                                         MIX                                                                            TIMES                                          STEP  REAGENTS AND OPERATIONS   MIN.                                           ______________________________________                                         1     CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                                  3                                              2     Methanol(MeOH) wash-30 ml. (2 times)                                                                     3                                              3     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                  3                                              4     50 percent TFA plus 5 percent 1,2-eth-                                                                   10                                                   anedithiol in CH.sub.2 CL.sub.2 --70 ml. (2 times)                       5     CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                                  3                                              6     TEA 12.5 percent in CH.sub.2 Cl.sub.2 --70 ml.                                                           5                                                    (2 times)                                                                7     MeOH wash-40 ml. (2 times)                                                                               2                                              8     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                  3                                              9     Boc-amino acid (10 mmoles) in 30 ml. of either                                                           30-300                                               DMF or CH.sub.2 Cl.sub.2, depending upon the                                   solubility of the particular protected amino                                   acid, (1 time) plus DCC (10 mmoles)                                            in CH.sub.2 Cl.sub.2                                                     10    MeOH wash-40 ml. (2 times)                                                                               3                                              11    TEA 12.5 percent in CH.sub.2 Cl.sub.2 --70 ml.                                                           3                                                    (1 time)                                                                 12    MeOH wash-30 ml. (2 times)                                                                               3                                              13    CH.sub.2 Cl.sub. 2 wash-80 ml. (2 times)                                                                 3                                              ______________________________________                                    

After step 13, if the syntheseis is performed manually, an aliquot is taken for a ninhydrin test: if the test is negative, go back to step 1 for coupling of the next amino acid; if the test is positive or slightly positive, go back and repeat steps 9 through 13.

The above schedule is used for coupling of each of the amino acids of the peptide of the invention after the first amino acid has been attached. N^(a) Boc protection is used for each of the remaining amino acids throughout the synthesis. N^(a) Boc-β-D-2NAL is prepared by a method known in the art, e.g. as described in detail in U.S. Pat. No. 4,234,571, issued Nov. 18, 1980. The side chain of Arg is protected with Tos. OBzl is used as a side chain protecting group for the hydroxyl group of Ser, and 2-6 dichlorobenzyl is used as the side chain protecting group for the hydroxyl group of D-Tyr. D-Trp is left unprotected. Boc-Arg(Tos) and Boc-D-Trp, which have low solubility in CH₂ C₂, are coupled using DMF:CH₂ Cl₂ mixtures.

After deblocking the a-amino group at the N-terminal, acetylation of the alpha-amino group of the N-terminal residue is achieved using a large excess of acetic anhydride in dichloromethane. The cleavage of the peptide from the resin and complete deprotection of the side chains takes place very readily at 0° C. with HF. Anisole is added as a scavenger prior to HF treatment. After the removal of HF under vacuum, the resin is extracted with 50% acetic acid, and the washings are lyophilized to provide a crude peptide powder.

Purification of the peptide is then effected by ion exchange chromatography on CMC (Whatman CM 32, using a gradient of 0.05 to 0.3M NH₄ OAc in 50/50 methanol/water) followed by partition chromatography in, a gel filtration column using the elution system: n-Butanol; 0.1N Acetic acid (1:1- volume ratio).

The peptide is judged to be homogeneous using thin layer chromatography and several different solvent systems, as well as by using reversed-phase high pressure liquid chromatography and an aqueous triethylammonium phosphate solution plus acetonitrile. Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared structure, showing substantially integer-values for each amino acid in the chain.

The peptides described hereinabove are tested in vivo to determine their effectiveness to prevent ovulation in female rats. In this test, a specified number of mature female Sprague-Dawley rats, i.e. ten, each having a body weight from 225 to 250 grams, is injected with from 10 micrograms to 1 microgram of peptide in corn oil at about noon on the day of proestrous. Proestrous is the afternoon before estrous (ovulation). A separate female rat group is used as a control to which the peptide is not administered. By comparing the number of the control female rats having ovulation at estrous to the number of the rats treated, which may also ovulate, evaluation of the effectiveness of the peptide as a contraceptive is made. Each of the peptides is considered to be significantly effective to prevent ovulation of female rats at a very low dosage, and each peptide is considered to be totally effective at a dose of about ten micrograms.

All of the peptides are considered to be effective to prevent ovulation of female mammans at very low dosages, some as low as about 10 micrograms per kilogram of body weight. These peptides may be administered in the form of pharmaceutically acceptable, nontoxic salts, such as acid addition salts, or of metal complexes, e.g., with zinc, barium, calcium magnesium, aluminun or the like (which are considered as addition salts for purposes of this application), or of combinations of the two. Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, nitrate, oxalate, fumarate, gluconate, tannate, maleate, acetate, citrate, benzoate, succinate, alginate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a pharmaceutically-acceptable diluent which includes a binder, such as tragacanth, corn starch or gelatin; a disintergrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used as part of the pharmaceutically-acceptable diluent, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.

The pharmaceutical compositions will usually contain the peptide in conjunction with a conventional,

The pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. Usually, the dosage will be from about 1 to about 100 micrograms of the peptide per kilogram of the body weight of the host when given intravenously; oral dosages will be higher. Overall, treatment of subjects with these peptides is generally carried out in the same manner as the clinical treatment using other antagonists of GnRH.

These peptides can be administered to mammals intravenously, subcutaneously, intramuscularly, orally, intranasally or intravaginally to achieve fertility inhibition and/or control and also in applications calling for reversible suppression of gonadal activity, such as for the management of precocious puberty or during radiation-or chemo-therapy. Effective dosages will vary with the form of administration and the particular species of mammal being treated. An example of one typical dosage form is a physiological saline solution containing the peptide which solution is administered to provide a dose in the range of about 0.1 to 2.5 mg/kg of body weight. Oral administration of the peptide may be given in either solid form or liquid form.

Although the invention has been described with regard to its preferred embodiments, it should be understood that changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims which are appended hereto. For example, other substitutions known in the art which do not significantly detract from the effectiveness of the peptides may be employed in the peptides of the invention. For instance, Gly-OCH₃, Gly-OCH₂ CH₃, Gly-NHNH₂ and Sar-NH₂ (Sar=sarcosine) are considered to be equivalents of the residues specified for R₁₀.

Particular features of the invention are emphasized in the claims that follow. 

What is claimed is:
 1. A peptide or a nontoxic salt thereof, said peptide having the formula: X-R₁ -R₂ -β-D-2NAL-Ser-Arg-R₆ -R7-Arg-Pro-R₁₀ wherein X is hydrogen, Acr or Ac; R₁ is β-D-2NAL, dehydroPro or 4Cl-D-Phe; R₂ is Cl-D-Phe, F-D-Phe, NO₂ -D-Phe, Br-D-Phe or C^(a) Me/Cl-D-Phe; R₆ is D-Tyr, C^(a) Me/Cl-D-Phe; R₆ is D-Tyr, β-D-2NAL or D-Arg; R₇ is Leu or NML; and R₁₀ is Gly-NH₂, NHCH₂ CH₃ or D-Ala-NH₂.
 2. A peptide in accordance with claim 1 wherein X is Ac and R₁ is β-D-2NAL.
 3. A peptide in accordance with claim 2 wherein R₇ is Leu.
 4. A peptide in accordance with claim 2 wherein R₂ is 4F-D-Phe.
 5. A peptide in accordance with claim 2 wherein R₆ is β-D-2NAL.
 6. A peptide in accordance with claim 2 wherein R₆ is D-Tyr.
 7. A peptide in accordance with claim 1 wherein R₁ is dehydroPro and R₆ is β-D-2NAL.
 8. A peptide in accordance with claim 7 wherein R₂ is 4F-D-Phe.
 9. A peptide in accordance with claim 8 wherein R₁₀ is D-Ala-NH₂.
 10. A peptide in accordance with claim 9 wherein R₇ is Leu.
 11. A peptide or a nontoxic salt thereof, which peptide has the formula: X-4Cl-D-Phe-R₂ -R₃ -Ser-Arg-R₆ -R₇ - Arg-Pro-R₁₀ wherein X is hydrogen, Acr or Ac; R₂ is Cl-D-Phe, F-D-Phe, NO₂ -D-Phe, Br-D-Phe or C^(a) Me-Cl-D-Phe; R₃ is D-Trp or β-D-2NAL; R₆ is D-Tyr, β-D-2NAL or D-Arg; R₇ is Leu or NML; and R₁₀ is Gly-NH₂, NHCH₂ CH₃ or D-Ala-NH₂.
 12. A peptide in accordance with claim 11 wherein R₃ is D-Trp, R₂ is 4Cl-D-Phe and R₇ is Leu.
 13. A peptide in accordance with claim 12 wherein R₆ is β-D-2NAL.
 14. A peptide in accordance with claim 13 wherein X is Ac and R₁₀ is D-Ala-NH₂.
 15. a peptide in accordance with claim 11 wherein R₃ is β-D-2NAL.
 16. A peptide or a nontoxic salt thereof, said peptide having the formula: X-dehydroPro-R₂ -R₃ -Ser-Arg-R₆ -Leu-Arg-Pro-R₁₀ wherein X is Ac, Acr or H, R₂ is 4Cl 4F-D-Phe, 4NO₂ -D-Phe, 4Br-D-Phe or C^(a) Me/4Cl-D-Phe; R₃ is β-D-2NAL or D-Trp; R₆ is D-Tyr, β-D-2NAL or D-Arg; and R₁₀ is Gly-NH₂, NHCH₂ CH₃ or D-Ala-NH₂.
 17. A peptide in accordance with claim 16 wherein R₂ is 4F-D-Phe and R₆ is β-D-2NAL.
 18. a peptide in accordance with claim 17 wherein X is Ac and R₁₀ is Gly-NH₂.
 19. A peptide in accordance with claim 16 wherein R₃ is β-D-2NAL.
 20. A peptide in accordance with claim 1 having the formula: Ac-dehydroPro-4F-D-Phe-β-D-2NAL-Ser-Arg-β-D-2NAL-Leu-Arg-Pro-Gly-NH₂. 